Plant regulatory elements and uses thereof

ABSTRACT

The invention provides recombinant DNA molecules and constructs, as well as their nucleotide sequences, useful for modulating gene expression in plants. The invention also provides transgenic plants, plant cells, plant parts, and seeds comprising the recombinant DNA molecules operably linked to heterologous transcribable DNA molecules, as are methods of their use.

REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. provisional application No.62/306,790, filed Mar. 11, 2016 which is herein incorporated byreference in its entirety.

INCORPORATION OF SEQUENCE LISTING

The sequence listing that is contained in the file named“MONS417US_sequencelisting.txt”, which is 30,143 bytes (as measured inMicrosoft Windows®) and was created on Mar. 8, 2017, is filed herewithby electronic submission, and is incorporated by reference herein.

FIELD OF THE INVENTION

The invention relates to the field of plant molecular biology; and plantgenetic engineering. More specifically, the invention relates to DNAmolecules useful for modulating gene expression in plants.

BACKGROUND

Regulatory elements are genetic elements that regulate gene activity bymodulating the transcription of an operably linked transcribable DNAmolecule. Such elements may include promoters, leaders, introns, and 3′untranslated regions and are useful in the field of plant molecularbiology and plant genetic engineering.

SUMMARY OF THE INVENTION

The invention provides novel gene regulatory elements for use in plants.The invention also provides DNA constructs comprising the regulatoryelements. The present invention also provides transgenic plant cells,plants, and seeds comprising the regulatory elements. In one embodiment,the regulatory elements are operably linked to a transcribable DNAmolecule. In certain embodiments, the transcribable DNA molecule may beheterologous with respect to the regulatory sequence. Thus, a regulatoryelement sequence provided by the invention may, in particularembodiments, be defined as operably linked to a heterologoustranscribable DNA molecule. The present invention also provides methodsof making and using the regulatory elements, the DNA constructscomprising the regulatory elements, and the transgenic plant cells,plants, and seeds comprising the regulatory elements operably linked toa transcribable DNA molecule.

Thus, in one aspect, the invention provides a recombinant DNA moleculecomprising a DNA sequence selected from the group consisting of: (a) asequence with at least about 85 percent sequence identity to any of SEQID NOs: 1-15; (b) a sequence comprising any of SEQ ID NOs: 1-15; and (c)a fragment of any of SEQ ID NOs: 1-15, wherein the fragment hasgene-regulatory activity; wherein the sequence is operably linked to aheterologous transcribable DNA molecule. By “heterologous transcribableDNA molecule,” it is meant that the transcribable DNA molecule isheterologous with respect to the polynucleotide sequence to which it isoperably linked. In specific embodiments, the recombinant DNA moleculecomprises a DNA sequence having at least about 90 percent, at least 91percent, at least 92 percent, at least 93 percent, at least 94 percent,at least 95 percent, at least 96 percent, at least 97 percent, at least98 percent, or at least 99 percent sequence identity to the DNA sequenceof any of SEQ ID NOs: 1-15. In particular embodiments, the DNA sequencecomprises a regulatory element. In some embodiments the regulatoryelement comprises a promoter. In still other embodiments, theheterologous transcribable DNA molecule comprises a gene of agronomicinterest, such as a gene capable of providing herbicide resistance inplants, or a gene capable of providing plant pest resistance in plants.In still other embodiments, the invention provides a constructcomprising a recombinant DNA molecule as provided herein.

In another aspect, provided herein are transgenic plant cells comprisinga recombinant DNA molecule comprising a DNA sequence selected from thegroup consisting of: (a) a sequence with at least about 85 percentsequence identity to any of SEQ ID NOs: 1-15; (b) a sequence comprisingany of SEQ ID NOs: 1-15; and (c) a fragment of any of SEQ ID NOs: 1-15,wherein the fragment has gene-regulatory activity; wherein the DNAsequence is operably linked to a heterologous transcribable DNAmolecule. In certain embodiments, the transgenic plant cell is amonocotyledonous plant cell. In other embodiments, the transgenic plantcell is a monocotyledonous plant cell or a dicotyledonous plant cell.

In still yet another aspect, further provided herein is a transgenicplant, or part thereof, comprising a recombinant DNA molecule comprisinga DNA sequence selected from the group consisting of: a) a sequence withat least 85 percent sequence identity to any of SEQ ID NOs: 1-15; b) asequence comprising any of SEQ ID NOs: 1-15; and c) a fragment of any ofSEQ ID NOs: 1-15, wherein the fragment has gene-regulatory activity;wherein the sequence is operably linked to a heterologous transcribableDNA molecule. In specific embodiments, the transgenic plant is a progenyplant of any generation that comprises the recombinant DNA molecule. Atransgenic seed comprising the recombinant DNA molecule that producessuch a transgenic plant when grown is also provided herein.

In another aspect, the invention provides a method of producing acommodity product comprising obtaining a transgenic plant or partthereof containing a recombinant DNA molecule of the invention andproducing the commodity product therefrom. In one embodiment, thecommodity product is processed seeds, grains, plant parts, oils andmeal.

In still yet another aspect, the invention provides a method ofproducing a transgenic plant comprising a recombinant DNA molecule ofthe invention comprising transforming a plant cell with the recombinantDNA molecule of the invention to produce a transformed plant cell andregenerating a transgenic plant from the transformed plant cell.

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO: 1 is a DNA sequence of a regulatory expression elements group(EXP) comprising a promoter derived from a Cucumis melo putativeFerredoxin 2 (Fe2) protein gene operably linked 5′ to its native leader.

SEQ ID NO: 2 is a promoter sequence derived from a Cucumis melo putativeFerredoxin 2 (Fe2) protein gene.

SEQ ID NO: 3 is a leader sequence derived from a Cucumis melo putativeFerredoxin 2 (Fe2) protein gene.

SEQ ID NO: 4 is a DNA sequence of an EXP comprising a promoter derivedfrom a Cucumis melo chlorophyll a-b binding protein 13 gene operablylinked 5′ to its native leader.

SEQ ID NO: 5 is a promoter sequence derived from a Cucumis melochlorophyll a-b binding protein 13 gene.

SEQ ID NO: 6 is a leader sequence derived from a Cucumis melochlorophyll a-b binding protein 13 gene.

SEQ ID NO: 7 is a DNA sequence of an EXP comprising a promoter derivedfrom a Cucumis melo B-box zinc finger protein 24-like gene operablylinked 5′ to its native leader.

SEQ ID NO: 8 is a promoter sequence derived from a Cucumis melo B-boxzinc finger protein 24-like gene.

SEQ ID NO: 9 is a leader sequence derived from a Cucumis melo B-box zincfinger protein 24-like gene.

SEQ ID NO: 10 is a DNA sequence of an EXP comprising a promoter derivedfrom a Medicago truncatula light harvesting complex protein b2 geneoperably linked 5′ to its native leader.

SEQ ID NO: 11 is a promoter sequence derived from a Medicago truncatulalight harvesting complex protein b2 gene.

SEQ ID NO: 12 is a leader sequence derived from a Medicago truncatulalight harvesting complex protein b2 gene.

SEQ ID NO: 13 is a DNA sequence of an EXP comprising a promoter derivedfrom a Medicago truncatula photosystem II chloroplast precursor geneoperably linked 5′ to its native leader.

SEQ ID NO: 14 is a promoter sequence derived from a Medicago truncatulaphotosystem II chloroplast precursor gene.

SEQ ID NO: 15 is a leader sequence promoter sequence derived from aMedicago truncatula photosystem II chloroplast precursor gene.

SEQ ID NO: 16 is an enhancer sequence derived from the promoter of theMedicago truncatula light harvesting complex protein b2 gene.

SEQ ID NO: 17 is a coding sequence for ß-glucuronidase (GUS) with aprocessable intron.

SEQ ID NO: 18 is a 3′ UTR sequence derived from the Gossypium barbadenseE6 gene.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides DNA molecules having gene-regulatory activity inplants. The nucleotide sequences of these DNA molecules are provided asSEQ ID NOs: 1-15. These DNA molecules are capable of affecting theexpression of an operably linked transcribable DNA molecule in planttissues, and therefore regulating gene expression of an operably linkedtransgene in transgenic plants. The invention also provides methods ofmodifying, producing, and using the same. The invention also providescompositions that include transgenic plant cells, plants, plant parts,and seeds containing the recombinant DNA molecules of the invention, andmethods for preparing and using the same.

The following definitions and methods are provided to better define thepresent invention and to guide those of ordinary skill in the art in thepractice of the present invention. Unless otherwise noted, terms are tobe understood according to conventional usage by those of ordinary skillin the relevant art.

DNA Molecules

As used herein, the term “DNA” or “DNA molecule” refers to adouble-stranded DNA molecule of genomic or synthetic origin, i.e., apolymer of deoxyribonucleotide bases or a DNA molecule, read from the 5′(upstream) end to the 3′ (downstream) end. As used herein, the term “DNAsequence” refers to the nucleotide sequence of a DNA molecule. Thenomenclature used herein corresponds to that of Title 37 of the UnitedStates Code of Federal Regulations § 1.822, and set forth in the tablesin WIPO Standard ST.25 (1998), Appendix 2, Tables 1 and 3.

As used herein, a “recombinant DNA molecule” is a DNA moleculecomprising a combination of DNA molecules that would not naturally occurtogether without human intervention. For instance, a recombinant DNAmolecule may be a DNA molecule that is comprised of at least two DNAmolecules heterologous with respect to each other, a DNA molecule thatcomprises a DNA sequence that deviates from DNA sequences that exist innature, or a DNA molecule that has been incorporated into a host cell'sDNA by genetic transformation or gene editing.

As used herein, the term “sequence identity” refers to the extent towhich two optimally aligned polynucleotide sequences or two optimallyaligned polypeptide sequences are identical. An optimal sequencealignment is created by manually aligning two sequences, e.g., areference sequence and another sequence, to maximize the number ofnucleotide matches in the sequence alignment with appropriate internalnucleotide insertions, deletions, or gaps. As used herein, the term“reference sequence” refers to a DNA sequence provided as to a DNAsequence provided as SEQ ID NOs: 1-15.

As used herein, the term “percent sequence identity” or “percentidentity” or “% identity” is the identity fraction multiplied by 100.The “identity fraction” for a sequence optimally aligned with areference sequence is the number of nucleotide matches in the optimalalignment, divided by the total number of nucleotides in the referencesequence, e.g., the total number of nucleotides in the full length ofthe entire reference sequence. Thus, one embodiment of the inventionprovides a DNA molecule comprising a sequence that, when optimallyaligned to a reference sequence, provided herein as SEQ ID NOs: 1-15,has at least about 85 percent identity, at least about 86 percentidentity, at least about 87 percent identity, at least about 88 percentidentity, at least about 89 percent identity, at least about 90 percentidentity, at least about 91 percent identity, at least about 92 percentidentity, at least about 93 percent identity, at least about 94 percentidentity, at least about 95 percent identity, at least about 96 percentidentity, at least about 97 percent identity, at least about 98 percentidentity, at least about 99 percent identity, or at least about 100percent identity to the reference sequence.

Regulatory Elements

Regulatory elements such as promoters, leaders (also known as 5′ UTRs),enhancers, introns, and transcription termination regions (or 3′ UTRs)play an integral part in the overall expression of genes in livingcells. The term “regulatory element,” as used herein, refers to a DNAmolecule having gene-regulatory activity. The term “gene-regulatoryactivity,” as used herein, refers to the ability to affect theexpression of an operably linked transcribable DNA molecule, forinstance by affecting the transcription and/or translation of theoperably linked transcribable DNA molecule. Regulatory elements, such aspromoters, leaders, enhancers, introns and 3′ UTRs that function inplants are therefore useful for modifying plant phenotypes throughgenetic engineering.

As used herein, a “regulatory expression element group” or “EXP”sequence may refer to a group of operably linked regulatory elements,such as enhancers, promoters, leaders, and introns. Thus, a regulatoryexpression element group may be comprised, for instance, of a promoteroperably linked 5′ to a leader sequence. EXP's useful in practicing thepresent invention include 1, 4, 7, 10, and 13.

Regulatory elements may be characterized by their gene expressionpattern, e.g., positive and/or negative effects such as constitutiveexpression or temporal, spatial, developmental, tissue, environmental,physiological, pathological, cell cycle, and/or chemically responsiveexpression, and any combination thereof, as well as by quantitative orqualitative indications. As used herein, a “gene expression pattern” isany pattern of transcription of an operably linked DNA molecule into atranscribed RNA molecule. The transcribed RNA molecule may be translatedto produce a protein molecule or may provide an antisense or otherregulatory RNA molecule, such as a double-stranded RNA (dsRNA), atransfer RNA (tRNA), a ribosomal RNA (rRNA), a microRNA (miRNA), and thelike.

As used herein, the term “protein expression” is any pattern oftranslation of a transcribed RNA molecule into a protein molecule.Protein expression may be characterized by its temporal, spatial,developmental, or morphological qualities, as well as by quantitative orqualitative indications.

A promoter is useful as a regulatory element for modulating theexpression of an operably linked transcribable DNA molecule. As usedherein, the term “promoter” refers generally to a DNA molecule that isinvolved in recognition and binding of RNA polymerase II and otherproteins, such as trans-acting transcription factors, to initiatetranscription. A promoter may be initially isolated from the 5′untranslated region (5′ UTR) of a genomic copy of a gene. Alternately,promoters may be synthetically produced or manipulated DNA molecules.Promoters may also be chimeric. Chimeric promoters are produced throughthe fusion of two or more heterologous DNA molecules. Promoters usefulin practicing the present invention include promoter elements comprisedwithin any of SEQ ID NOs: 2, 5, 8, 11, and 14, or fragments or variantsthereof. In specific embodiments of the invention, the claimed DNAmolecules and any variants or derivatives thereof as described herein,are further defined as comprising promoter activity, i.e., are capableof acting as a promoter in a host cell, such as in a transgenic plant.In still further specific embodiments, a fragment may be defined asexhibiting promoter activity possessed by the starting promoter moleculefrom which it is derived, or a fragment may comprise a “minimalpromoter” which provides a basal level of transcription and is comprisedof a TATA box, other known transcription factor binding site motif, orequivalent DNA sequence for recognition and binding of the RNApolymerase II complex for initiation of transcription.

In one embodiment, fragments of a promoter sequence disclosed herein areprovided. Promoter fragments may comprise promoter activity, asdescribed above, and may be useful alone or in combination with otherpromoters and promoter fragments, such as in constructing chimericpromoters, or in combination with other EXPs and EXP fragments. Inspecific embodiments, fragments of a promoter are provided comprising atleast about 50, at least about 75, at least about 95, at least about100, at least about 125, at least about 150, at least about 175, atleast about 200, at least about 225, at least about 250, at least about275, at least about 300, at least about 500, at least about 600, atleast about 700, at least about 750, at least about 800, at least about900, or at least about 1000 contiguous nucleotides, or longer, of a DNAmolecule having promoter activity as disclosed herein. In certainembodiments, the invention provides fragments of any one of SEQ ID NOs:1-15, having the activity of the full length sequence. Methods forproducing such fragments from a starting promoter molecule are wellknown in the art.

Compositions derived from any of the promoter elements comprised withinany of SEQ ID NOs: 2, 5, 8, 11, and 14, such as internal or 5′deletions, for example, can be produced using methods known in the artto improve or alter expression, including by removing elements that haveeither positive or negative effects on expression; duplicating elementsthat have positive or negative effects on expression; and/or duplicatingor removing elements that have tissue- or cell-specific effects onexpression. Compositions derived from any of the promoter elementscomprised within any of SEQ ID NOs: 2, 5, 8, 11, and 14 comprised of 3′deletions in which the TATA box element or equivalent sequence thereofand downstream sequence is removed can be used, for example, to makeenhancer elements. Further deletions can be made to remove any elementsthat have positive or negative; tissue-specific; cell-specific; ortiming-specific (such as, but not limited to, circadian rhythm) effectson expression. Any of the promoter elements comprised within any of SEQID NOs: 2, 5, 8, 11, and 14 and fragments or enhancers derived therefromcan be used to make chimeric transcriptional regulatory elementcompositions.

In accordance with the invention, a promoter or promoter fragment may beanalyzed for the presence of known promoter elements, i.e., DNA sequencecharacteristics, such as a TATA box and other known transcription factorbinding site motifs. Identification of such known promoter elements maybe used by one of skill in the art to design variants of the promoterhaving a similar expression pattern to the original promoter.

As used herein, the term “leader” refers to a DNA molecule isolated fromthe untranslated 5′ region (5′ UTR) a gene and defined generally as anucleotide segment between the transcription start site (TSS) and theprotein coding sequence start site. Alternately, leaders may besynthetically produced or manipulated DNA elements. A leader can be usedas a 5′ regulatory element for modulating expression of an operablylinked transcribable DNA molecule. Leader molecules may be used with aheterologous promoter or with their native promoter. Leaders useful inpracticing the present invention include SEQ ID NOs: 3, 6, 9, 12, and 15or any of the leader elements comprised within any of SEQ ID NOs: 1, 4,7, 10, and 13 or fragments or variants thereof. In specific embodiments,such DNA sequences may be defined as being capable of acting as a leaderin a host cell, including, for example, a transgenic plant cell. In oneembodiment, such sequences are decoded as comprising leader activity.

The leader sequences (also referred to as 5′ UTRs) presented as SEQ IDNOs: 3, 6, 9, 12, and 15 or any of the leader elements comprised withinany of SEQ ID NOs: 1, 4, 7, 10, and 13 may be comprised of regulatoryelements, or may adopt secondary structures that can have an effect ontranscription or translation of an operably linked transcribable DNAmolecule. The leader sequences presented as SEQ ID NOs: 3, 6, 9, 12, and15 or any of the leader elements comprised within any of SEQ ID NOs: 1,4, 7, 10, and 13 can be used in accordance with the invention to makechimeric regulatory elements that affect transcription or translation ofa an operably linked transcribable DNA molecule.

As used herein, the term “intron” refers to a DNA molecule that may beisolated or identified from a gene and may be defined generally as aregion spliced out during messenger RNA (mRNA) processing prior totranslation. Alternately, an intron may be a synthetically produced ormanipulated DNA element. An intron may contain enhancer elements thateffect the transcription of operably linked genes. An intron may be usedas a regulatory element for modulating expression of an operably linkedtranscribable DNA molecule. A construct may comprise an intron, and theintron may or may not be heterologous with respect to the transcribableDNA molecule. Examples of introns in the art include the rice actinintron and the corn HSP70 intron.

In plants, the inclusion of some introns in gene constructs leads toincreased mRNA and protein accumulation relative to constructs lackingthe intron. This effect has been termed “intron mediated enhancement”(IME) of gene expression. Introns known to stimulate expression inplants have been identified in maize genes (e.g., tubA1, Adh1, Sh1, andUbi1), in rice genes (e.g., tpi) and in dicotyledonous plant genes likethose from petunia (e.g., rbcS), potato (e.g., st-ls1) and fromArabidopsis thaliana (e.g., ubq3 and pat1). It has been shown thatdeletions or mutations within the splice sites of an intron reduce geneexpression, indicating that splicing might be needed for IME. However,IME in dicotyledonous plants has been shown by point mutations withinthe splice sites of the pat1 gene from A. thaliana. Multiple uses of thesame intron in one plant have been shown to exhibit disadvantages. Inthose cases, it is necessary to have a collection of basic controlelements for the construction of appropriate recombinant DNA elements.

As used herein, the terms “3′ transcription termination molecule,” “3′untranslated region” or “3′ UTR” refer to a DNA molecule that is usedduring transcription to the untranslated region of the 3′ portion of anmRNA molecule. The 3′ untranslated region of an mRNA molecule may begenerated by specific cleavage and 3′ polyadenylation, also known as apolyA tail. A 3′ UTR may be operably linked to and located downstream ofa transcribable DNA molecule and may include a polyadenylation signaland other regulatory signals capable of affecting transcription, mRNAprocessing, or gene expression. PolyA tails are thought to function inmRNA stability and in initiation of translation. Examples of 3′transcription termination molecules in the art are the nopaline synthase3′ region; wheat hsp17 3′ region, pea rubisco small subunit 3′ region,cotton E6 3′ region, and the coixin 3′ UTR.

3′ UTRs typically find beneficial use for the recombinant expression ofspecific DNA molecules. A weak 3′ UTR has the potential to generateread-through, which may affect the expression of the DNA moleculelocated in the neighboring expression cassettes. Appropriate control oftranscription termination can prevent read-through into DNA sequences(e.g., other expression cassettes) localized downstream and can furtherallow efficient recycling of RNA polymerase to improve gene expression.Efficient termination of transcription (release of RNA Polymerase IIfrom the DNA) is prerequisite for re-initiation of transcription andthereby directly affects the overall transcript level. Subsequent totranscription termination, the mature mRNA is released from the site ofsynthesis and template transported to the cytoplasm. Eukaryotic mRNAsare accumulated as poly(A) forms in vivo, making it difficult to detecttranscriptional termination sites by conventional methods. However,prediction of functional and efficient 3′ UTRs by bioinformatics methodsis difficult in that there are no conserved DNA sequences that wouldallow easy prediction of an effective 3′ UTR.

From a practical standpoint, it is typically beneficial that a 3′ UTRused in an expression cassette possesses the following characteristics.The 3′ UTR should be able to efficiently and effectively terminatetranscription of the transgene and prevent read-through of thetranscript into any neighboring DNA sequence, which can be comprised ofanother expression cassette as in the case of multiple expressioncassettes residing in one transfer DNA (T-DNA), or the neighboringchromosomal DNA into which the T-DNA has inserted. The 3′ UTR should notcause a reduction in the transcriptional activity imparted by thepromoter, leader, enhancers, and introns that are used to driveexpression of the DNA molecule. In plant biotechnology, the 3′ UTR isoften used for priming of amplification reactions of reverse transcribedRNA extracted from the transformed plant and used to: (1) assess thetranscriptional activity or expression of the expression cassette onceintegrated into the plant chromosome; (2) assess the copy number ofinsertions within the plant DNA; and (3) assess zygosity of theresulting seed after breeding. The 3′ UTR is also used in amplificationreactions of DNA extracted from the transformed plant to characterizethe intactness of the inserted cassette.

As used herein, the term “enhancer” or “enhancer element” refers to acis-acting regulatory element, a.k.a. cis-element, which confers anaspect of the overall expression pattern, but is usually insufficientalone to drive transcription, of an operably linked transcribable DNAmolecule. Unlike promoters, enhancer elements do not usually include atranscription start site (TSS) or TATA box or equivalent DNA sequence. Apromoter or promoter fragment may naturally comprise one or moreenhancer elements that affect the transcription of an operably linkedDNA sequence. An enhancer element may also be fused to a promoter toproduce a chimeric promoter cis-element, which confers an aspect of theoverall modulation of gene expression. An example of an enhancer elementderived from the Medicago truncatula light harvesting complex protein b2precursor gene promoter is provided as SEQ ID NO: 16.

Many promoter enhancer elements are believed to bind DNA-bindingproteins and/or affect DNA topology, producing local conformations thatselectively allow or restrict access of RNA polymerase to the DNAtemplate or that facilitate selective opening of the double helix at thesite of transcriptional initiation. An enhancer element may function tobind transcription factors that regulate transcription. Some enhancerelements bind more than one transcription factor, and transcriptionfactors may interact with different affinities with more than oneenhancer domain. Enhancer elements can be identified by a number oftechniques, including deletion analysis, i.e., deleting one or morenucleotides from the 5′ end or internal to a promoter; DNA bindingprotein analysis using DNase I footprinting, methylation interference,electrophoresis mobility-shift assays, in vivo genomic footprinting byligation-mediated polymerase chain reaction (PCR), and otherconventional assays; or by DNA sequence similarity analysis using knowncis-element motifs or enhancer elements as a target sequence or targetmotif with conventional DNA sequence comparison methods, such as BLAST.The fine structure of an enhancer domain can be further studied bymutagenesis (or substitution) of one or more nucleotides or by otherconventional methods known in the art. Enhancer elements can be obtainedby chemical synthesis or by isolation from regulatory elements thatinclude such elements, and they can be synthesized with additionalflanking nucleotides that contain useful restriction enzyme sites tofacilitate subsequence manipulation. Thus, the design, construction, anduse of enhancer elements according to the methods disclosed herein formodulating the expression of operably linked transcribable DNA moleculesare encompassed by the invention.

As used herein, the term “chimeric” refers to a single DNA moleculeproduced by fusing a first DNA molecule to a second DNA molecule, whereneither the first nor the second DNA molecule would normally be found inthat configuration, i.e. fused to the other. The chimeric DNA moleculeis thus a new DNA molecule not otherwise normally found in nature. Asused herein, the term “chimeric promoter” refers to a promoter producedthrough such manipulation of DNA molecules. A chimeric promoter maycombine two or more DNA fragments; for example, the fusion of a promoterto an enhancer element. Thus, the design, construction, and use ofchimeric promoters according to the methods disclosed herein formodulating the expression of operably linked transcribable DNA moleculesare encompassed by the present invention.

Chimeric regulatory elements can be designed to comprise variousconstituent elements which may be operatively linked by various methodsknown in the art, such as restriction enzyme digestion and ligation,ligation independent cloning, modular assembly of PCR products duringamplification, or direct chemical synthesis of the regulatory element,as well as other methods known in the art. The resulting variouschimeric regulatory elements can be comprised of the same, or variantsof the same, constituent elements but differ in the DNA sequence or DNAsequences that comprise the linking DNA sequence or sequences that allowthe constituent parts to be operatively linked. In the invention, a DNAsequence provided as SEQ ID NOs: 1-15 may provide a regulatory elementreference sequence, wherein the constituent elements that comprise thereference sequence may be joined by methods known in the art and maycomprise substitutions, deletions, and/or insertions of one or morenucleotides or mutations that naturally occur in bacterial and plantcell transformation.

As used herein, the term “variant” refers to a second DNA molecule, suchas a regulatory element, that is in composition similar, but notidentical to, a first DNA molecule, and wherein the second DNA moleculestill maintains the general functionality, i.e. the same or similarexpression pattern, for instance through more or less equivalenttranscriptional activity, of the first DNA molecule. A variant may be ashorter or truncated version of the first DNA molecule and/or an alteredversion of the sequence of the first DNA molecule, such as one withdifferent restriction enzyme sites and/or internal deletions,substitutions, and/or insertions. A “variant” can also encompass aregulatory element having a nucleotide sequence comprising asubstitution, deletion, and/or insertion of one or more nucleotides of areference sequence, wherein the derivative regulatory element has moreor less or equivalent transcriptional or translational activity than thecorresponding parent regulatory molecule. Regulatory element “variants”will also encompass variants arising from mutations that naturally occurin bacterial and plant cell transformation. In the present invention, apolynucleotide sequence provided as SEQ ID NOs: 1-15 may be used tocreate variants that are in similar in composition, but not identicalto, the DNA sequence of the original regulatory element, while stillmaintaining the general functionality, i.e., the same or similarexpression pattern, of the original regulatory element. Production ofsuch variants of the invention is well within the ordinary skill of theart in light of the disclosure and is encompassed within the scope ofthe invention.

Reference in this application to an “isolated DNA molecule”, or anequivalent term or phrase, is intended to mean that the DNA molecule isone that is present alone or in combination with other compositions, butnot within its natural environment. For example, nucleic acid elementssuch as a coding sequence, intron sequence, untranslated leadersequence, promoter sequence, transcriptional termination sequence, andthe like, that are naturally found within the DNA of the genome of anorganism are not considered to be “isolated” so long as the element iswithin the genome of the organism and at the location within the genomein which it is naturally found. However, each of these elements, andsubparts of these elements, would be “isolated” within the scope of thisdisclosure so long as the element is not within the genome of theorganism and at the location within the genome in which it is naturallyfound. For the purposes of this disclosure, any transgenic nucleotidesequence, i.e., the nucleotide sequence of the DNA inserted into thegenome of the cells of a plant or bacterium, or present in anextrachromosomal vector, would be considered to be an isolatednucleotide sequence whether it is present within the plasmid or similarstructure used to transform the cells, within the genome of the plant orbacterium, or present in detectable amounts in tissues, progeny,biological samples or commodity products derived from the plant orbacterium.

The efficacy of the modifications, duplications, or deletions describedherein on the desired expression aspects of a particular transgene maybe tested empirically in stable and transient plant assays, such asthose described in the working examples herein, so as to validate theresults, which may vary depending upon the changes made and the goal ofthe change in the starting DNA molecule.

Constructs

As used herein, the term “construct” means any recombinant DNA moleculesuch as a plasmid, cosmid, virus, phage, or linear or circular DNA orRNA molecule, derived from any source, capable of genomic integration orautonomous replication, comprising a DNA molecule where at least one DNAmolecule has been linked to another DNA molecule in a functionallyoperative manner, i.e. operably linked. As used herein, the term“vector” means any construct that may be used for the purpose oftransformation, i.e., the introduction of heterologous DNA or RNA into ahost cell. A construct typically includes one or more expressioncassettes. As used herein, an “expression cassette” refers to a DNAmolecule comprising at least a transcribable DNA molecule operablylinked to one or more regulatory elements, typically at least a promoterand a 3′ UTR.

As used herein, the term “operably linked” refers to a first DNAmolecule joined to a second DNA molecule, wherein the first and secondDNA molecules are so arranged that the first DNA molecule affects thefunction of the second DNA molecule. The two DNA molecules may or maynot be part of a single contiguous DNA molecule and may or may not beadjacent. For example, a promoter is operably linked to a transcribableDNA molecule if the promoter modulates transcription of thetranscribable DNA molecule of interest in a cell. A leader, for example,is operably linked to DNA sequence when it is capable of affecting thetranscription or translation of the DNA sequence.

The constructs of the invention may be provided, in one embodiment, asdouble tumor-inducing (Ti) plasmid border constructs that have the rightborder (RB or AGRtu.RB) and left border (LB or AGRtu.LB) regions of theTi plasmid isolated from Agrobacterium tumefaciens comprising a T-DNAthat, along with transfer molecules provided by the A. tumefacienscells, permit the integration of the T-DNA into the genome of a plantcell (see, e.g., U.S. Pat. No. 6,603,061). The constructs may alsocontain the plasmid backbone DNA segments that provide replicationfunction and antibiotic selection in bacterial cells, e.g., anEscherichia coli origin of replication such as ori322, a broad hostrange origin of replication such as oriV or oriRi, and a coding regionfor a selectable marker such as Spec/Strp that encodes for Tn7aminoglycoside adenyltransferase (aadA) conferring resistance tospectinomycin or streptomycin, or a gentamicin (Gm, Gent) selectablemarker gene. For plant transformation, the host bacterial strain isoften A. tumefaciens ABI, C58, or LBA4404; however, other strains knownto those skilled in the art of plant transformation can function in theinvention.

Methods are known in the art for assembling and introducing constructsinto a cell in such a manner that the transcribable DNA molecule istranscribed into a functional mRNA molecule that is translated andexpressed as a protein. For the practice of the invention, conventionalcompositions and methods for preparing and using constructs and hostcells are well known to one skilled in the art. Typical vectors usefulfor expression of nucleic acids in higher plants are well known in theart and include vectors derived from the Ti plasmid of Agrobacteriumtumefaciens and the pCaMVCN transfer control vector.

Various regulatory elements may be included in a construct, includingany of those provided herein. Any such regulatory elements may beprovided in combination with other regulatory elements. Suchcombinations can be designed or modified to produce desirable regulatoryfeatures. In one embodiment, constructs of the invention comprise atleast one regulatory element operably linked to a transcribable DNAmolecule operably linked to a 3′ UTR.

Constructs of the invention may include any promoter or leader providedherein or known in the art. For example, a promoter of the invention maybe operably linked to a heterologous non-translated 5′ leader such asone derived from a heat shock protein gene. Alternatively, a leader ofthe invention may be operably linked to a heterologous promoter such asthe Cauliflower Mosaic Virus 35S transcript promoter.

Expression cassettes may also include a transit peptide coding sequencethat encodes a peptide that is useful for sub-cellular targeting of anoperably linked protein, particularly to a chloroplast, leucoplast, orother plastid organelle; mitochondria; peroxisome; vacuole; or anextracellular location. Many chloroplast-localized proteins areexpressed from nuclear genes as precursors and are targeted to thechloroplast by a chloroplast transit peptide (CTP). Examples of suchisolated chloroplast proteins include, but are not limited to, thoseassociated with the small subunit (SSU) of ribulose-1,5,-bisphosphatecarboxylase, ferredoxin, ferredoxin oxidoreductase, the light-harvestingcomplex protein I and protein II, thioredoxin F, and enolpyruvylshikimate phosphate synthase (EPSPS). Chloroplast transit peptides aredescribed, for example, in U.S. Pat. No. 7,193,133. It has beendemonstrated that non-chloroplast proteins may be targeted to thechloroplast by the expression of a heterologous CTP operably linked tothe transgene encoding a non-chloroplast proteins.

Transcribable DNA Molecules

As used herein, the term “transcribable DNA molecule” refers to any DNAmolecule capable of being transcribed into a RNA molecule, including,but not limited to, those having protein coding sequences and thoseproducing RNA molecules having sequences useful for gene suppression.The type of DNA molecule can include, but is not limited to, a DNAmolecule from the same plant, a DNA molecule from another plant, a DNAmolecule from a different organism, or a synthetic DNA molecule, such asa DNA molecule containing an antisense message of a gene, or a DNAmolecule encoding an artificial, synthetic, or otherwise modifiedversion of a transgene. Exemplary transcribable DNA molecules forincorporation into constructs of the invention include, e.g., DNAmolecules or genes from a species other than the species into which theDNA molecule is incorporated or genes that originate from, or arepresent in, the same species, but are incorporated into recipient cellsby genetic engineering methods rather than classical breedingtechniques.

A “transgene” refers to a transcribable DNA molecule heterologous to ahost cell at least with respect to its location in the host cell genomeand/or a transcribable DNA molecule artificially incorporated into ahost cell's genome in the current or any prior generation of the cell.

A regulatory element, such as a promoter of the invention, may beoperably linked to a transcribable DNA molecule that is heterologouswith respect to the regulatory element. As used herein, the term“heterologous” refers to the combination of two or more DNA moleculeswhen such a combination is not normally found in nature. For example,the two DNA molecules may be derived from different species and/or thetwo DNA molecules may be derived from different genes, e.g., differentgenes from the same species or the same genes from different species. Aregulatory element is thus heterologous with respect to an operablylinked transcribable DNA molecule if such a combination is not normallyfound in nature, i.e., the transcribable DNA molecule does not naturallyoccur operably linked to the regulatory element.

The transcribable DNA molecule may generally be any DNA molecule forwhich expression of a transcript is desired. Such expression of atranscript may result in translation of the resulting mRNA molecule, andthus protein expression. Alternatively, for example, a transcribable DNAmolecule may be designed to ultimately cause decreased expression of aspecific gene or protein. In one embodiment, this may be accomplished byusing a transcribable DNA molecule that is oriented in the antisensedirection. One of ordinary skill in the art is familiar with using suchantisense technology. Any gene may be negatively regulated in thismanner, and, in one embodiment, a transcribable DNA molecule may bedesigned for suppression of a specific gene through expression of adsRNA, siRNA or miRNA molecule.

Thus, one embodiment of the invention is a recombinant DNA moleculecomprising a regulatory element of the invention, such as those providedas SEQ ID NOs: 1-15, operably linked to a heterologous transcribable DNAmolecule so as to modulate transcription of the transcribable DNAmolecule at a desired level or in a desired pattern when the constructis integrated in the genome of a transgenic plant cell. In oneembodiment, the transcribable DNA molecule comprises a protein-codingregion of a gene and in another embodiment the transcribable DNAmolecule comprises an antisense region of a gene.

Genes of Agronomic Interest

A transcribable DNA molecule may be a gene of agronomic interest. Asused herein, the term “gene of agronomic interest” refers to atranscribable DNA molecule that, when expressed in a particular planttissue, cell, or cell type, confers a desirable characteristic. Theproduct of a gene of agronomic interest may act within the plant inorder to cause an effect upon the plant morphology, physiology, growth,development, yield, grain composition, nutritional profile, disease orpest resistance, and/or environmental or chemical tolerance or may actas a pesticidal agent in the diet of a pest that feeds on the plant. Inone embodiment of the invention, a regulatory element of the inventionis incorporated into a construct such that the regulatory element isoperably linked to a transcribable DNA molecule that is a gene ofagronomic interest. In a transgenic plant containing such a construct,the expression of the gene of agronomic interest can confer a beneficialagronomic trait. A beneficial agronomic trait may include, for example,but is not limited to, herbicide tolerance, insect control, modifiedyield, disease resistance, pathogen resistance, modified plant growthand development, modified starch content, modified oil content, modifiedfatty acid content, modified protein content, modified fruit ripening,enhanced animal and human nutrition, biopolymer productions,environmental stress resistance, pharmaceutical peptides, improvedprocessing qualities, improved flavor, hybrid seed production utility,improved fiber production, and desirable biofuel production.

Examples of genes of agronomic interest known in the art include thosefor herbicide resistance (U.S. Pat. Nos. 6,803,501; 6,448,476;6,248,876; 6,225,114; 6,107,549; 5,866,775; 5,804,425; 5,633,435; and5,463,175), increased yield (U.S. Pat. Nos. RE38,446; 6,716,474;6,663,906; 6,476,295; 6,441,277; 6,423,828; 6,399,330; 6,372,211;6,235,971; 6,222,098; and 5,716,837), insect control (U.S. Pat. Nos.6,809,078; 6,713,063; 6,686,452; 6,657,046; 6,645,497; 6,642,030;6,639,054; 6,620,988; 6,593,293; 6,555,655; 6,538,109; 6,537,756;6,521,442; 6,501,009; 6,468,523; 6,326,351; 6,313,378; 6,284,949;6,281,016; 6,248,536; 6,242,241; 6,221,649; 6,177,615; 6,156,573;6,153,814; 6,110,464; 6,093,695; 6,063,756; 6,063,597; 6,023,013;5,959,091; 5,942,664; 5,942,658, 5,880,275; 5,763,245; and 5,763,241),fungal disease resistance (U.S. Pat. Nos. 6,653,280; 6,573,361;6,506,962; 6,316,407; 6,215,048; 5,516,671; 5,773,696; 6,121,436;6,316,407; and 6,506,962), virus resistance (U.S. Pat. Nos. 6,617,496;6,608,241; 6,015,940; 6,013,864; 5,850,023; and 5,304,730), nematoderesistance (U.S. Pat. No. 6,228,992), bacterial disease resistance (U.S.Pat. No. 5,516,671), plant growth and development (U.S. Pat. Nos.6,723,897 and 6,518,488), starch production (U.S. Pat. Nos. 6,538,181;6,538,179; 6,538,178; 5,750,876; 6,476,295), modified oils production(U.S. Pat. Nos. 6,444,876; 6,426,447; and 6,380,462), high oilproduction (U.S. Pat. Nos. 6,495,739; 5,608,149; 6,483,008; and6,476,295), modified fatty acid content (U.S. Pat. Nos. 6,828,475;6,822,141; 6,770,465; 6,706,950; 6,660,849; 6,596,538; 6,589,767;6,537,750; 6,489,461; and 6,459,018), high protein production (U.S. Pat.No. 6,380,466), fruit ripening (U.S. Pat. No. 5,512,466), enhancedanimal and human nutrition (U.S. Pat. Nos. 6,723,837; 6,653,530;6,5412,59; 5,985,605; and 6,171,640), biopolymers (U.S. Pat. Nos.RE37,543; 6,228,623; and U.S. Pat. Nos. 5,958,745, and 6,946,588),environmental stress resistance (U.S. Pat. No. 6,072,103),pharmaceutical peptides and secretable peptides (U.S. Pat. Nos.6,812,379; 6,774,283; 6,140,075; and 6,080,560), improved processingtraits (U.S. Pat. No. 6,476,295), improved digestibility (U.S. Pat. No.6,531,648) low raffinose (U.S. Pat. No. 6,166,292), industrial enzymeproduction (U.S. Pat. No. 5,543,576), improved flavor (U.S. Pat. No.6,011,199), nitrogen fixation (U.S. Pat. No. 5,229,114), hybrid seedproduction (U.S. Pat. No. 5,689,041), fiber production (U.S. Pat. Nos.6,576,818; 6,271,443; 5,981,834; and 5,869,720) and biofuel production(U.S. Pat. No. 5,998,700).

Alternatively, a gene of agronomic interest can affect the abovementioned plant characteristics or phenotypes by encoding a RNA moleculethat causes the targeted modulation of gene expression of an endogenousgene, for example by antisense (see, e.g. U.S. Pat. No. 5,107,065);inhibitory RNA (“RNAi,” including modulation of gene expression bymiRNA-, siRNA-, trans-acting siRNA-, and phased sRNA-mediatedmechanisms, e.g., as described in published applications U.S.2006/0200878 and U.S. 2008/0066206, and in U.S. patent application Ser.No. 11/974,469); or cosuppression-mediated mechanisms. The RNA couldalso be a catalytic RNA molecule (e.g., a ribozyme or a riboswitch; see,e.g., U.S. 2006/0200878) engineered to cleave a desired endogenous mRNAproduct. Methods are known in the art for constructing and introducingconstructs into a cell in such a manner that the transcribable DNAmolecule is transcribed into a molecule that is capable of causing genesuppression.

Selectable Markers

Selectable marker transgenes may also be used with the regulatoryelements of the invention. As used herein the term “selectable markertransgene” refers to any transcribable DNA molecule whose expression ina transgenic plant, tissue or cell, or lack thereof, can be screened foror scored in some way. Selectable marker genes, and their associatedselection and screening techniques, for use in the practice of theinvention are known in the art and include, but are not limited to,transcribable DNA molecules encoding ß-glucuronidase (GUS), greenfluorescent protein (GFP), proteins that confer antibiotic resistance,and proteins that confer herbicide tolerance. An example of a selectablemarker transgene is provided as SEQ ID NO:17.

Cell Transformation

The invention is also directed to a method of producing transformedcells and plants that comprise one or more regulatory elements operablylinked to a transcribable DNA molecule.

The term “transformation” refers to the introduction of a DNA moleculeinto a recipient host. As used herein, the term “host” refers tobacteria, fungi, or plants, including any cells, tissues, organs, orprogeny of the bacteria, fungi, or plants. Plant tissues and cells ofparticular interest include protoplasts, calli, roots, tubers, seeds,stems, leaves, seedlings, embryos, and pollen.

As used herein, the term “transformed” refers to a cell, tissue, organ,or organism into which a foreign DNA molecule, such as a construct, hasbeen introduced. The introduced DNA molecule may be integrated into thegenomic DNA of the recipient cell, tissue, organ, or organism such thatthe introduced DNA molecule is inherited by subsequent progeny. A“transgenic” or “transformed” cell or organism may also include progenyof the cell or organism and progeny produced from a breeding programemploying such a transgenic organism as a parent in a cross andexhibiting an altered phenotype resulting from the presence of a foreignDNA molecule. The introduced DNA molecule may also be transientlyintroduced into the recipient cell such that the introduced DNA moleculeis not inherited by subsequent progeny. The term “transgenic” refers toa bacterium, fungus, or plant containing one or more heterologous DNAmolecules.

There are many methods well known to those of skill in the art forintroducing DNA molecules into plant cells. The process generallycomprises the steps of selecting a suitable host cell, transforming thehost cell with a vector, and obtaining the transformed host cell.Methods and materials for transforming plant cells by introducing aplant construct into a plant genome in the practice of this inventioncan include any of the well-known and demonstrated methods. Suitablemethods include, but are not limited to, bacterial infection (e.g.,Agrobacterium), binary BAC vectors, direct delivery of DNA (e.g., byPEG-mediated transformation, desiccation/inhibition-mediated DNA uptake,electroporation, agitation with silicon carbide fibers, and accelerationof DNA coated particles), gene editing (e.g., CRISPR-Cas systems), amongothers.

Host cells may be any cell or organism, such as a plant cell, algalcell, algae, fungal cell, fungi, bacterial cell, or insect cell. Inspecific embodiments, the host cells and transformed cells may includecells from crop plants.

A transgenic plant subsequently may be regenerated from a transgenicplant cell of the invention. Using conventional breeding techniques orself-pollination, seed may be produced from this transgenic plant. Suchseed, and the resulting progeny plant grown from such seed, will containthe recombinant DNA molecule of the invention, and therefore will betransgenic.

Transgenic plants of the invention can be self-pollinated to provideseed for homozygous transgenic plants of the invention (homozygous forthe recombinant DNA molecule) or crossed with non-transgenic plants ordifferent transgenic plants to provide seed for heterozygous transgenicplants of the invention (heterozygous for the recombinant DNA molecule).Both such homozygous and heterozygous transgenic plants are referred toherein as “progeny plants.” Progeny plants are transgenic plantsdescended from the original transgenic plant and containing therecombinant DNA molecule of the invention. Seeds produced using atransgenic plant of the invention can be harvested and used to growgenerations of transgenic plants, i.e., progeny plants of the invention,comprising the construct of this invention and expressing a gene ofagronomic interest. Descriptions of breeding methods that are commonlyused for different crops can be found in one of several reference books,see, e.g., Allard, Principles of Plant Breeding, John Wiley & Sons, NY,U. of CA, Davis, Calif., 50-98 (1960); Simmonds, Principles of CropImprovement, Longman, Inc., NY, 369-399 (1979); Sneep and Hendriksen,Plant breeding Perspectives, Wageningen (ed), Center for AgriculturalPublishing and Documentation (1979); Fehr, Soybeans: Improvement,Production and Uses, 2nd Edition, Monograph, 16:249 (1987); Fehr,Principles of Variety Development, Theory and Technique, (Vol. 1) andCrop Species Soybean (Vol. 2), Iowa State Univ., Macmillan Pub. Co., NY,360-376 (1987).

The transformed plants may be analyzed for the presence of the gene orgenes of interest and the expression level and/or profile conferred bythe regulatory elements of the invention. Those of skill in the art areaware of the numerous methods available for the analysis of transformedplants. For example, methods for plant analysis include, but are notlimited to, Southern blots or northern blots, PCR-based approaches,biochemical analyses, phenotypic screening methods, field evaluations,and immunodiagnostic assays. The expression of a transcribable DNAmolecule can be measured using TaqMan® (Applied Biosystems, Foster City,Calif.) reagents and methods as described by the manufacturer and PCRcycle times determined using the TaqMan® Testing Matrix. Alternatively,the Invader® (Third Wave Technologies, Madison, Wis.) reagents andmethods as described by the manufacturer can be used to evaluatetransgene expression.

The invention also provides for parts of a plant of the invention. Plantparts include, but are not limited to, leaves, stems, roots, tubers,seeds, endosperm, ovule, and pollen. Plant parts of the invention may beviable, nonviable, regenerable, and/or non-regenerable. The inventionalso includes and provides transformed plant cells comprising a DNAmolecule of the invention. The transformed or transgenic plant cells ofthe invention include regenerable and/or non-regenerable plant cells.

The invention also provides a commodity product that is produced from atransgenic plant or part thereof containing the recombinant DNA moleculeof the invention. Commodity products of the invention contain adetectable amount of DNA comprising a DNA sequence selected from thegroup consisting of SEQ ID NOs: 1-15. As used herein, a “commodityproduct” refers to any composition or product which is comprised ofmaterial derived from a transgenic plant, seed, plant cell, or plantpart containing the recombinant DNA molecule of the invention. Commodityproducts include but are not limited to processed seeds, grains, plantparts, and meal. A commodity product of the invention will contain adetectable amount of DNA corresponding to the recombinant DNA moleculeof the invention. Detection of one or more of this DNA in a sample maybe used for determining the content or the source of the commodityproduct. Any standard method of detection for DNA molecules may be used,including methods of detection disclosed herein.

The invention may be more readily understood through reference to thefollowing examples, which are provided by way of illustration, and arenot intended to be limiting of the invention, unless specified. Itshould be appreciated by those of skill in the art that the techniquesdisclosed in the following examples represent techniques discovered bythe inventors to function well in the practice of the invention.However, those of skill in the art should, in light of the presentdisclosure, appreciate that many changes can be made in the specificembodiments that are disclosed and still obtain a like or similar resultwithout departing from the spirit and scope of the invention, thereforeall matter set forth or shown in the accompanying drawings is to beinterpreted as illustrative and not in a limiting sense.

EXAMPLES Example 1 Identification and Cloning of Regulatory Elements

Novel transcriptional regulatory elements and regulatory expressionelement groups (EXPs) were identified and cloned from genomic DNA of thedicot species Cucumis melo (WSH-39-1070AN) and Medicago truncatula.

Transcriptional regulatory elements were selected based upon proprietaryand public microarray data derived from transcriptional profilingexperiments conducted in soybean (Glycine max) and Arabidopsis, as wellas homology based searches using known dicot sequences as queriesagainst proprietary Cucumis melo and proprietary and public Medicagotruncatula sequences.

Using the identified sequences, a bioinformatic analysis was conductedto identify regulatory elements within the amplified DNA. For example,bioinformatics analysis was performed to identify the transcriptionalstart site (TSS) and any bi-directionality, introns, or upstream codingsequence present in the sequence. Using the results of this analysis,regulatory elements were defined within the DNA sequences and primersdesigned to amplify the regulatory elements. The corresponding DNAmolecule for each regulatory element was amplified using standardpolymerase chain reaction conditions with primers containing uniquerestriction enzyme sites and genomic DNA isolated from Cucumis melo andMedicago truncatula. The resulting DNA fragments were ligated into baseplant expression vectors using standard restriction enzyme digestion ofcompatible restriction sites and DNA ligation methods.

Analysis of the regulatory element TSS and intron/exon splice junctionscan be performed using transformed plant tissue. Briefly, the plants aretransformed with the plant expression vectors comprising the cloned DNAfragments operably linked to a heterologous transcribable DNA molecule.Next, the 5′ RACE System for Rapid Amplification of cDNA Ends, Version2.0 (Invitrogen, Carlsbad, Calif. 92008) is used to confirm theregulatory element TSS and intron/exon splice junctions by analyzing theDNA sequence of the produced mRNA transcripts.

The DNA sequences encoding the Cucumis and Medicago transcriptionalregulatory expression element groups or EXP sequences which arecomprised of a promoter element, operably linked to a leader element arepresented in Table 1 along with their corresponding promoters andleaders.

TABLE 1 Transcriptional regulatory expression element groups, promoters,leaders and introns isolated from Cucumis melo and Medicago truncatulaSEQ Description ID NO: Gene Annotation EXP-CUCme.Fe2:1 1 PutativeFerredoxin 2 (Fe2) protein P-CUCme.Fe2:1 2 Putative Ferredoxin 2 (Fe2)protein L-CUCme.Fe2:1 3 Putative Ferredoxin 2 (Fe2) proteinEXP-CUCme.CipLhcb:1 4 Chlorophyll a-b binding protein 13P-CUCme.CipLhcb:1 5 Chlorophyll a-b binding protein 13 L-CUCme.CipLhcb:16 Chlorophyll a-b binding protein 13 EXP-CUCme.Bbz:1 7 B-box zinc fingerprotein 24-like P-CUCme.Bbz:1 8 B-box zinc finger protein 24-likeL-CUCme.Bbz:1 9 B-box zinc finger protein 24-like EXP-Mt.Lhcb2:1:1 10Light harvesting complex protein b2 P-Mt.Lhcb2-1:2:1 11 Light harvestingcomplex protein b2 L-Mt.Lhcb2-1:2:1 12 Light harvesting complex proteinb2 EXP-Mt.PSII-T:1:1 13 Photosystem II chloroplast precursorP-Mt.PSII-T-1:2:1 14 Photosystem II chloroplast precursorL-Mt.PSII-T-1:2:1 15 Photosystem II chloroplast precursor

Example 2 Analysis of Regulatory Elements Driving GUS Expression inStably Transformed Soybean Plants

Soybean plants were transformed with vectors, specifically plantexpression vectors, containing a test regulatory element drivingexpression of the ß-glucuronidase (GUS) transgene. The resulting plantswere analyzed for GUS protein expression, to assess the effect of theselected regulatory elements on expression.

Soybean plants were transformed with the plant GUS expression constructslisted in Table 2. The regulatory elements were cloned into a base plantexpression vector using standard methods known in the art. The resultingplant expression vectors contained a right border region fromAgrobacterium tumefaciens (B-AGRtu.right border), a first transgeneselection cassette used for selection of transformed plant cells thatconfers either resistance to either the herbicide glyphosate or theantibiotic, spectinomycin; a second transgene cassette to assess theactivity of the regulatory element, which comprised an EXP sequenceoperably linked 5′ to a coding sequence for ß-glucuronidase (GUS,GOI-Ec.uidA+St.LS1:1:1, SEQ ID NO: 17) containing a processable intronderived from the potato light-inducible tissue-specific ST-LS1 gene(Genbank Accession: X04753), operably linked 5′ to a 3′ terminationregion from the Gossypium barbadense E6 gene (T-Gb.E6-3b:3b:1, SEQ IDNO: 18); and a left border region from Agrobacterium tumefaciens(B-AGRtu.left border).

TABLE 2 Regulatory elements and corresponding GUS expression plasmidconstructs SEQ ID Construct EXP Description NO: pMON142244EXP-CUCme.Fe2:1 1 pMON142241 EXP-CUCme.CipLhcb:1 4 pMON142216EXP-CUCme.Bbz:1 7 pMON116798 EXP-Mt.Lhcb2:1:1 10 pMON116792EXP-Mt.PSII-T:1:1 13

Soybean plant cells were transformed using the binary transformationvector constructs described above by Agrobacterium-mediatedtransformation, using methods known in the art. The resultingtransformed plant cells were induced to form whole soybean plants.

Histochemical GUS analysis was used for qualitative and quantitativeexpression analysis of transformed plants. Whole tissue sections wereincubated with GUS staining solution X-Gluc(5-bromo-4-chloro-3-indolyl-b-glucuronide) (1 milligram/milliliter) foran appropriate length of time, rinsed, and visually inspected for bluecoloration. GUS activity was qualitatively determined by direct visualinspection or inspection under a microscope using selected plant organsand tissues.

For quantitative analysis of GUS expression, total protein was extractedfrom selected tissues of transformed soybean plants. One microgram oftotal protein was used with the fluorogenic substrate4-methylumbelliferyl-β-D-glucuronide (MUG) in a total reaction volume of50 microliters. The reaction product, 4-methylumbelliferone (4-MU), ismaximally fluorescent at high pH, where the hydroxyl group is ionized.Addition of a basic solution of sodium carbonate simultaneously stopsthe assay and adjusts the pH for quantifying the fluorescent product.Fluorescence was measured with excitation at 365 nm, emission at 445 nmusing a Fluoromax-3 with Micromax Reader, with slit width set atexcitation 2 nm and emission 3 nm. Values are provided in units of nmolGUS/hour/mg total protein.

The following tissues were sampled for GUS expression in the R₀generation; Vn5 stage sink leaf, source leaf, and root; R1 stagepetiole, source leaf, flower, and cotyledon; R3 stage pod and immatureseed; and yellow pod stage embryo and cotyledon. Tables 3 and 4 belowshow the mean quantitative GUS expression for each of the sampledtissues driven by the regulatory elements presented in Table 2.

TABLE 3 Mean quantitative GUS expression in stably transformed soybeanplants driven by regulatory elements derived from Cucumis melo StageOrgan EXP-CUCme.Fe2:1 EXP-CUCme.CipLhcb:1 EXP-CUCme.Bbz:1 Vn5 Sink Leaf391 159 100 Vn5 Root 40 35 24 Vn5 Source Leaf 431 120 185 R1 Petiole 581724 32 R1 Source Leaf 600 357 41 R1 Flower 290 111 30 R5 Cotyledon 38 3113 R3 Pod 778 1199 85 R3 Immature Seed 52 56 47 Yellow Pod Embryo 23 2838 Yellow Pod Cotyledon 33 35 49

TABLE 4 Mean quantitative GUS expression in stably transformed soybeanplants driven by regulatory elements derived from Medicago truncatulaStage Organ EXP-Mt.Lhcb2:1:1 EXP-Mt.PSII-T:1:1 Vn5 Sink Leaf 108 22 Vn5Root 21 11 Vn5 Source Leaf 148 42 R1 Petiole 153 83 R1 Source Leaf 94118 R1 Flower 39 21 R5 Cotyledon 13 9 R3 Pod 85 33 R3 Immature Seed 5 9Yellow Embryo 4 0 Pod Yellow Cotyledon 4 0 Pod

As can be seen in Tables 3 and 4, each of the regulatory elements has aunique pattern of expression in the tissues sampled. BothEXP-CUCme.Fe2:1 (SEQ ID NO: 1) and EXP-CUCme.CipLhcb:1 (SEQ ID NO: 4)express highly in the R3 pod and show the lowest level of expression inthe R3 immature seed, Vn5 root, R5 cotyledon, and the yellow pod stageembryo and cotyledon. GUS expression driven by EXP-CUCme.Fe2:1 was alsohigh in the Vn5 stage sink and source leaf and R1 stage petiole, sourceleaf, and flower. The regulatory element EXP-CUCme.Bbz:1 (SEQ ID NO: 7)demonstrated highest expression in the Vn5 stage sink and source leafand R3 stage pod. GUS expression driven by EXP-Mt.Lhcb2:1:1 (SEQ ID NO:10) was highest in the Vn5 stage source leaf and R1 petiole.EXP-Mt.PSII-T:1:1 (SEQ ID NO: 13) demonstrated highest expression in theR1 stage source leaf.

Each regulatory element provides a unique pattern of expression whichcan be used to optimally drive expression of different transgenes,depending on the desired tissue preference for expression.

Example 3 Enhancer Elements Derived from the Regulatory Elements

Enhancers are derived from the promoter elements presented as SEQ IDNOs: 2, 5, 8, 11, and 14. The enhancer element may be comprised of oneor more cis regulatory elements that when operably linked 5′ or 3′ to apromoter element, or operably linked 5′ or 3′ to additional enhancerelements that are operably linked to a promoter, can enhance or modulateexpression levels of a transcribable DNA molecule, or provide expressionof a transcribable DNA molecule in a specific cell type or plant organor at a particular time point in development or circadian rhythm.Enhancers are made by removing the TATA box or functionally similarelements and any downstream sequence from the promoters that allowtranscription to be initiated from a promoter sequence, for example thesequences presented as SEQ ID NOs: 2, 5, 8, 11, and 14 or fragmentsthereof.

The TATA box in plant promoters is not as highly conserved as in someother Eukaryotic organisms. Therefore, in order to define a fragment asan enhancer, one first must identify the transcriptional start site(TSS) of the gene, wherein the 5′ UTR is first transcribed. For example,the transcriptional regulatory element, EXP-Mt.Lhcb2:1:1 (SEQ ID NO: 10)is comprised of the promoter element, P-Mt.Lhcb2-1:2:1 (SEQ ID NO: 11),operably linked to the 5′ UTR or leader element, L-Mt.Lhcb2-1:2:1 (SEQID NO: 12). Within the 1837 bp promoter element, P-Mt.Lhcb2-1:2:1 (SEQID NO: 11), the putative core TATA-like element is found withinnucleotides 1798 through 1803. An enhancer fragment derived from theP-Mt.Lhcb2-1:2:1 could comprise nucleotides 1 through 1797 of SEQ ID NO:11, resulting in the sequence presented as SEQ ID NO: 16 (E-Mt.Lhcb2).Enhancers derived from the promoter, P-Mt.Lhcb2-1:2:1 (SEQ ID NO: 11)can further comprise smaller fragments of E-Mt.Lhcb2 (SEQ ID NO: 16).The effectiveness of the enhancer elements derived from the promoter,P-Mt.Lhcb2-1:2:1 (SEQ ID NO: 11) is empirically determined by building achimeric transcriptional regulatory element comprising an enhancerderived from the promoter, P-Mt.Lhcb2-1:2:1 (SEQ ID NO: 11), which isoperably linked to a promoter and leader and used to drive expression ofa transcribable DNA molecule such as GUS in stable or transient plantassay.

Further refinement of the enhancer element may be required and isvalidated empirically. In addition, position of the enhancer elementrelative to other elements within a chimeric transcriptional regulatoryelement is also empirically determined, since the order of each elementwithin the chimeric transcriptional regulatory element may impartdifferent effects, depending upon the relative positions of eachelement. Some promoter elements will have multiple TATA box or TATAbox-like elements and potentially multiple transcription start sites.Under those circumstances, it may be necessary to first identify wherethe first TSS is located and then begin designing enhancers using thefirst TSS to prevent the potential initiation of transcription fromoccurring within a putative enhancer element.

Enhancer elements, derived from the promoter elements presented as SEQID NOs: 2, 5, 8, 11, and 14 are cloned using methods known in the art tobe operably linked 5′ or 3′ to a promoter element, or operably linked 5′or 3′ to additional enhancer elements that are operably linked to apromoter. Alternatively, enhancer elements can be cloned, using methodsknown in the art, to provide a larger enhancer element that is comprisedof two or more copies of the enhancer and cloned using methods known inthe art to be operably linked 5′ or 3′ to a promoter element, oroperably linked 5′ or 3′ to additional enhancer elements that areoperably linked to a promoter producing a chimeric transcriptionalregulatory element. Enhancer elements can also be cloned using methodsknown in the art to be operably linked 5′ to a promoter element derivedfrom a different genus organism, or operably linked 5′ or 3′ toadditional enhancer elements derived from other genus organisms that areoperably linked to a promoter derived from either the same or differentgenus organism, resulting in a chimeric transcriptional regulatoryelement. A GUS expression plant transformation vector may be constructedusing methods known in the art similar to the constructs described inExample 2 above in which the resulting plant expression vectors containa right border region from Agrobacterium tumefaciens (B-AGRtu.rightborder), a first transgene selection cassette used for selection oftransformed plant cells that confers resistance to the antibiotic,spectinomycin; and a second transgene cassette to test the enhancerelement comprised of, the enhancer element operably linked 5′ or 3′ to apromoter element or operably linked 5′ or 3′ to additional enhancerelements that are in turn operably linked to a promoter which isoperably linked 5′ to a leader element, operably linked to a codingsequence for ß-glucuronidase (GUS, GOI-Ec.uidA+St.LS1:1:1, SEQ ID NO:17) containing a processable intron derived from the potatolight-inducible tissue-specific ST-LS1 gene (Genbank Accession: X04753),operably linked to a 3′ termination region from the Gossypium barbadenseE6 gene (T-Gb.E6-3b:3b:1, SEQ ID NO: 18), and a left border region fromA. tumefaciens (B-AGRtu.left border). The resulting plasmids are used totransform soybean plants or other genus plants by the methods describedabove. Alternatively, protoplast cells derived from soybean or othergenus plants are transformed using methods known in the art to performtransient assays

GUS expression driven by a regulatory element comprising one or moreenhancers is evaluated in stable or transient plant assays to determinethe effects of the enhancer element on expression of a transcribable DNAmolecule. Modifications to one or more enhancer elements or duplicationof one or more enhancer elements may be performed based upon empiricalexperimentation and the resulting gene expression regulation that isobserved using each regulatory element composition. Altering therelative positions of one or more enhancers in the resulting regulatoryor chimeric regulatory elements may affect the transcriptional activityor specificity of the regulatory or chimeric regulatory element and isdetermined empirically to identify the best enhancers for the desiredtransgene expression profile within the corn plant or other genus plant.

Having illustrated and described the principles of the presentinvention, it should be apparent to persons skilled in the art that theinvention can be modified in arrangement and detail without departingfrom such principles. We claim all modifications that are within thespirit and scope of the claims. All publications and published patentdocuments cited herein are hereby incorporated by reference to the sameextent as if each individual publication or patent application isspecifically and individually indicated to be incorporated by reference.

What is claimed is:
 1. A recombinant DNA molecule comprising apolynucleotide selected from the group consisting of: a) apolynucleotide with at least 95 percent sequence identity to SEQ ID NO:5, wherein the polynucleotide has promoter activity; b) a polynucleotidewith at least 97 percent sequence identity to SEQ ID NO: 4, wherein thepolynucleotide has gene-regulatory activity; c) a polynucleotidecomprising SEQ ID NO: 4 or 5; and d) a fragment comprising at least 150contiguous nucleotides of SEQ ID NO: 5, wherein the fragment haspromoter activity; wherein said DNA molecule is operably linked to aheterologous transcribable DNA molecule.
 2. The recombinant DNA moleculeof claim 1, wherein said polynucleotide has at least 97 percent sequenceidentity to SEQ ID NO:
 5. 3. The recombinant DNA molecule of claim 2,wherein said polynucleotide has at least 99 percent sequence identity toSEQ ID NO: 4 or
 5. 4. The recombinant DNA molecule of claim 3, whereinsaid polynucleotide comprises the DNA sequence of SEQ ID NO: 4 or
 5. 5.The recombinant DNA molecule of claim 1, wherein the heterologoustranscribable DNA molecule comprises a gene of agronomic interest. 6.The recombinant DNA molecule of claim 5, wherein the gene of agronomicinterest confers herbicide tolerance in plants.
 7. The recombinant DNAmolecule of claim 5, wherein the gene of agronomic interest confers pestresistance in plants.
 8. A transgenic plant cell comprising arecombinant DNA molecule comprising a polynucleotide selected from thegroup consisting of: a) a polynucleotide with at least 95 percentsequence identity to SEQ ID NO: 5, wherein the polynucleotide haspromoter activity; b) a polynucleotide with at least 97 percent sequenceidentity to SEQ ID NO: 4, wherein the polynucleotide has gene-regulatoryactivity; c) a polynucleotide comprising SEQ ID NO: 4 or 5; and d) afragment comprising at least 150 contiguous nucleotides of SEQ ID NO: 5,wherein the fragment has promoter activity; wherein said DNA molecule isoperably linked to a heterologous transcribable DNA molecule.
 9. Thetransgenic plant cell of claim 8, wherein said transgenic plant cell isa monocotyledonous plant cell.
 10. The transgenic plant cell of claim 8,wherein said transgenic plant cell is a dicotyledonous plant cell.
 11. Atransgenic plant, or part thereof, comprising the recombinant DNAmolecule of claim
 1. 12. A progeny plant of the transgenic plant ofclaim 11, or a part thereof, wherein the progeny plant or part thereofcomprises said recombinant DNA molecule.
 13. A transgenic seed, whereinthe seed comprises the recombinant DNA molecule of claim
 1. 14. A methodof producing a commodity product comprising obtaining the transgenicplant or part thereof according to claim 11 and producing the commodityproduct therefrom.
 15. The method of claim 14, wherein the commodityproduct is protein concentrate, protein isolate, grain, starch, seeds,meal, flour, biomass, or seed oil.
 16. A method of expressing atranscribable DNA molecule comprising obtaining the transgenic plantaccording to claim 11 and cultivating the plant, wherein thetranscribable DNA is expressed.